Project summary/abstract The prevalence of autoimmune and allergic diseases is rising. The human antibody isotype IgE plays a central role in the pathogenesis of allergic disease by mediating antigen dependent signals through the IgE Fc- receptors (Fc?RI and Fc?RII). Fc?RI, the high affinity IgE Fc-receptor, binds IgE very tightly (KD <1nmol/L) As a result, mast cells and basophils expressing Fc?RI circulate preloaded with IgE:Fc?RI complexes, poised for activation and degranulation upon antigen encounter. Activation of these cells can induce clinical outcomes ranging from allergic symptoms to anaphylaxis and death. Omalizumab, an anti-IgE therapeutic monoclonal antibody, has validated IgE as a therapeutic target for allergic disorders. Omalizumab primarily acts as a competitive inhibitor of IgE:Fc?RI interactions, and binds free circulating IgE, often taking weeks to achieve a therapeutic response. In contrast, we have now selected and characterized two Designed Ankyrin Repeat Protein (DARPin) inhibitors, named E2_79 and bi53_79, that are ~1-3 orders of magnitude more efficient than omalizumab at immediately disrupting IgE receptor complexes and suppressing the activation of allergic effector cells. The DARPin bi53_79 is also approximately an order of magnitude more potent than omalizumab at preventing passive cutaneous anaphylaxis in vivo. These new disruptive inhibitors of preformed IgE:Fc?RI complexes are not limited by the slow dissociation of IgE from Fc?RI, and can theoretically disarm mast cells and basophils on contact. Yet, the viability of DARPin based therapies is not clear. The work in this proposal aims to define the mechanism of the disruptive inhibitors E2_79 and bi53_79, and apply that knowledge to design or identify therapeutically viable antibody-based disruptive inhibitors of IgE:Fc?RI complexes. These agents would serve as a basis for the design of new, more potent anti-IgE drugs, that could potentially be used to suppress acute allergic responses.